Patent Application
20110136146
The present invention relates to bioassays for mutated polyQ protein associated with disease and their use as diagnostic tools, for monitoring disease progression or for monitoring the efficacy of treatment of the disease.
A number of diseases are associated with the expression of proteins with polyglutamine repeats, such as Huntington's Disease (HD), spinal bulbar muscular atrophy, several spinocerebellar ataxias and dentatorubral-pallidoluysian atrophy. These disorders are collectively termed ‘polyglutamine diseases’. HD is the most common inherited neurodegenerative disorder with a prevalence of 5 to 8 cases per 100′000. Its main clinical manifestations include motoric dysfunction, psychiatric disturbances and dementia. Numerous symptomatic treatments have been tried for HD without any substantial success (Bonelli and Wenning, 2006) and no approved treatments for HD exist (Bates, 2003). HD is the founding member of the polyglutamine (polyQ) disease family composed of nine autosomal-dominant inherited disorders whose common characteristic is a polyQ-repeat expansion in different ubiquitously expressed proteins (Ross, 2002). The genetic mutation causing HD is a polyglutamine expansion in the huntingtin protein. Expansions beyond 36 glutamines become pathogenic and appear to affect protein folding and successive formation of toxic intracellular fragments and aggregates. The expanded polyQ repeat in the huntingtin gene (Htt) lies in exon 1 and leads to the expression of mutant polyQ-Htt protein (Gusella et al., 1983). The polyQ-repeat expansion may promote a conversion from a native random-coiled to a cylindrical, parallel beta-sheet conformation tethered by hydrogen bonds between the polyglutamine strands (Perutz et al., 1994). Similar to other neurodegenerative disease characterized by protein misfolding like Alzheimer's Disease or Parkinson's Disease, the proteins with helical beta-sheet conformation are prone to form non-soluble protein aggregates (Benzinger et al., 2000).
HD-like symptoms are reversed when expression of mutant Htt is down-regulated in the brain of HD mouse models by RNA interference (DiFiglia et al., 2007) or by tetracyclin-regulated conditional expression (Yamamoto et al., 2000). Interestingly, mutant polyQ- and wild type-Htt are differently metabolized by the cell and display a different pattern of posttranslational modifications (phosphorylation (Warby et al., 2005), proteolytic cleavage (Gafni et al., 2004; Graham et al., 2006; Wellington et al., 2002), cellular localization (Davies et al., 1997; van Roon-Mom et al., 2002) and degradation (Ravikumar et al., 2002). These findings prompted discovery work for HD therapeutics aimed at influencing the misfolding or the clearance of mutant Htt e.g. through upregulation of the chaperone system or induction of the autophagy degradation pathway (Yamamoto et al., 2006). Such approaches may find application for other neurodegenerative diseases caused by protein misfolding.
Currently, there is no bioassay available to assess the effect of such therapies on mutant polyglutamine proteins, e.g. mutant Htt levels in e.g. clinical trials or in therapy.
The present invention relates to bioassays for soluble mutated polyQ protein associated with disease and their use as diagnostic tools, for monitoring disease progression or for monitoring the efficacy of treatment of the disease.
In a preferred embodiment the bioassay used is a new homogenous time resolved Förster resonance energy transfer method for Htt detection suitable for high-throughput screening in a neuronal cell line. We show that this “time resolved Förster resonance energy transfer immunoassay” (in the following “Bioassay”) can be modified to quantify endogenous, full-length soluble polyQ-Htt in cellular, animal and human samples. The use of such a bioassay allows for the use of soluble mutant Htt as a marker for disease progression or to monitor the efficacy of drug treatments in preclinical and clinical trials as well as in therapeutic treatment. As the design of the method is highly flexible, it is applicable for use in connection with other diseases, especially diseases associated with other members of the polyQ-family. These proteins are typically expressed intracellularly and assays useful for diagnosis and/or monitoring of disease progression/therapy have not been available until now.
In a preferred embodiment of the present invention the bioassay measures the soluble forms of the polyQ protein, e.g. polyQ huntingtin. In another preferred embodiment of the present invention the bioassay measures the aggregated form of the polyQ protein, e.g. polyQ huntingtin. In yet another preferred embodiment of the present invention the bioassay measures both the soluble and the aggregated form of the polyQ protein, e.g. polyQ huntingtin.
Thus, in one aspect the present invention provides an immunoassay for measuring the amount of the mutated form (polyQ form) of a protein in a biological sample, wherein the protein is selected from the group of huntingtin, androgen receptor, atrophin 1, ataxin 1, ataxin 2, ataxin 3, ataxin 7, TATA box binding protein or alpha1a voltage dependent calcium channel subunit.
In a preferred embodiment of the immunoassay according to the present invention the absolute or relative amount of the corresponding wild-type protein in the sample is additionally measured in the immunoassay.
In a further preferred embodiment of the immunoassay according to the present invention the extent of post-translational modifications of the mutated protein is additionally measured, such as cellular modifications of the expressed protein such as fragmentation by e.g proteolytical cleavage, phosphorylation, acetylation, ubiquitination, SUMOylation, lipid modification or other covalent modifications of the polypeptide backbone.
In a further preferred embodiment of the immunoassay according to the present invention, the immunoassay is a single step assay, i.e. an immunoassay in which no separation or washing is necessary and which can preferably be run after a single biochemical handling. In a further preferred embodiment of the immunoassay according to the present invention, the immunoassay detection technology is based on time-resolved Förster resonance energy transfer or electrochemiluminescence.
In a further preferred embodiment of the immunoassay according to the present invention, the immunoassay detection technology is time-resolved Förster resonance energy transfer. In a further preferred embodiment of the immunoassay according to the present invention, the immunoassay comprises the following steps.
In a further preferred embodiment of the immunoassay according to the present invention, the absolute or relative amount of the corresponding wild-type protein in the sample is additionally measured in the biological sample by additionally contacting it with a third antibody specific for the wild-type form of the protein and labeled with a different fluorophore suited for detecting the lanthanide emitted signal.
In a further preferred embodiment of the immunoassay according to the present invention, the polyQ-protein is polyQ-huntingtin.
In a further preferred embodiment of the immunoassay according to the present invention, the biological sample is derived from the brain, from blood, from muscle or heart or derived from any other peripheral tissue such as skin or hair.
In another aspect of the invention an immunoassay is used for measuring the amount of the mutated polyQ form of a protein in a biological sample, wherein the protein is selected from the group of huntingtin, androgen receptor, atrophin 1, ataxin 1, ataxin 2, ataxin 3, ataxin 7, TATA box binding protein or alphala voltage dependent calcium channel subunit; as a diagnostic tool, for monitoring disease progression or for monitoring the efficacy of treatment of the disease associated with the mutated polyQ form of the protein. The immunoassay is preferably an immunoassay as described in the present application.
A. Amino acid sequences of peptides analyzed with the antibody pair beta1 (β1) & 25H10 are shown with the respective epitopes in bold letters. B. Time-resolved FRET analysis of peptides (3 ng/well in duplicates) shown in A. The H1 sequence was selected for tagging mutant Htt and the I6 sequence was selected for tagging wild-type Htt. C. Schemes of cDNA constructs expressed in the inducible HN10 cell lines, indicated are also the antibody binding sites of 25H10, 32A7 and beta1 (β1) at the C-terminal tag. 2B7 is specific for an endogenous amino-terminal epitope of Htt. D. Schematic representation of the protocol used for the cell-based time-resolved FRET assay.
A. Western blot analysis of induced expression of wild-type (573-Q25) and mutant (573-Q72) Htt in HN10 cells compared to that of endogenous full-length Htt. B. After 3 days of induction, the HN10 cell line transfected with mutant and wild-type Htt shows stable Htt expression over time and multiple cell passages. C. Sensitive and specific detection of wild-type or mutant Htt by time-resolved FRET using the indicated antibody combinations after 3 days induction (average value, n=3, standard deviation). D. Optimization of cell lysis conditions for detection of mutant Htt573-Q72 in cells induced for 3 days utilizing the 2B7 & β1 antibody pair (time-resolved FRET measured 50 min after antibody addition, n=6, standard deviation). E. The Z′-factor remained stable between 35 and 110 min after cell lysis and addition of the detecting antibodies although the absolute time-resolved FRET signal increased over time (not shown), induction for 3 days of 573-Q72 HN10 cells (n=6, standard deviation). F. Dose-dependent increase in mutant Htt573-Q72 expression in cells treated with different inducer concentrations (n=6, standard deviation, EC50 ˜250 nM). A-C. the data were generated in 96-well plate format. D-F. the data were generated in 1536-well plate format.
Monoclonal antibodies labeled for time-resolved FRET are added by a single pipetting step to cell lysates or tissue homogenates. Simultaneous binding to Htt brings the two antibodies in proximity thus enabling energy transfer from the europium cryptate to the D2-fluorophore. The unique long-lived fluorescence emitted by the lanthanoide cryptate allows for time-resolved measurement and thus temporal distinction from interfering short-lived background fluorescence. As a consequence, accurate Htt determination is possible in strongly colored and autofluorescent biological samples such as blood. B. Scheme of the antibody binding sites on human Htt (tagged Htt573 fragment). 2B7 and MW1 are specific for Htt epitopes located at the amino-terminus and on the polyQ repeat of Htt, respectively. The three monoclonal antibodies Beta1, 32A7 and 25H10 are specific for epitopes added to the carboxy-terminus of the tagged Htt573 fragment. MW1 binding to the polyQ-repeat results in stronger and increased binding to Htt as a function of the polyQ-length (represented by the two antibodies displayed in the figure).
A: Time-resolved FRET detection of tagged wild-type (25Q) and mutant (72Q) Htt 573 fragments in induced HN10 cell lysates using the Htt antibody pair 2B7 & MW1. Lysates prepared from non-induced cells served as negative controls. B: Specific detection of induced untagged Htt exon1 fragments in HN10 cells by time-resolved FRET with 2B7 & MW1. C: Quantification of standard amounts of purified recombinant wild-type (25Q) or mutant (46Q) Htt 573 protein by time-resolved FRET with 2B7 & MW1. D. Lentiviral-mediated expression of untagged wild-type (25Q) and mutant (72Q) Htt 548 fragments in ESC-derived neurons analyzed by time-resolved FRET with 2B7 & MW1 revealed that the Htt signal increased as function of polyQ-length although similar Htt expression levels were shown by western blot with 2B7. Htt fragments were not detected in mock infected cells. Tubulin was the loading control. E: Specific detection of endogenous Htt in 140Q-knock-in ESCs. Htt knock-out ESCs (KO) served as negative control. Murine wild-type Htt with 7Q was not detected by 2B7 & MW1. F: Analysis of cell lysates obtained from knock-in ESC-derived neurons expressing endogenous Htt with increasing polyQ-length confirms the polyQ-dependency of the time-resolved FRET assay for Htt. Expression of Htt in the cell lysates shown in this Figure were all confirmed independently by western blot analysis (not shown). All panels show average values of n=3 with error bars indicating standard deviation values.
Determination of levels of expression of tamed wild-type and mutant Htt573 in the same sample. HN10 cells expressing both wild-type Htt (25Q) and mutant Htt (72Q) truncated at amino acid 573 and tagged with the beta1 and 32A7 or with beta1 and 25H10 antigens, respectively. Beta1 carried the terbium cryptate mojety that transferred the fluorescent enrgy to 25H10 labelled with D2 as well as to 32A7 labelled with Alexa488. D2 and Alexa488 signals were measured in two different wave length channels.
SDS poly-acrylamide gel electrophoresis of purified wild-type (Htt573Q25) and mutant (Htt573Q46) protein produced in basteria. Defined amounts of bovine serum albumin (BSA) were loded as a control to estimate the amount of purified Htt obtained in the purification method applied.
Defined amount of recombinat wild-type Htt (25Q) and mutant Htt (46) were analyzed by duplex time resolved Förster resonance energy transfer using the indicated antibody pairs. Whilst 2B7 & MW1 (Alexa488 channel) recognized better mutant Htt in a polyQ-dependent manner, 2B7 & 4C9 (D2 channel) resulted in a stronger signal for wild-type Htt than for the mutant Htt protein with elongated polyQ.
Defined amount of recombinat wild-type Htt (25Q) and mutant Htt (46) were analyzed by duplex time resolved Förster resonance energy transfer using the indicated antibody pairs. Whilst 2B7 & MW1 (Alexa488 channel) recognized better mutant Htt in a polyQ-dependent manner, 2B7 & 2166 (D2 channel) resulted in a stronger signal for wild-type Htt than for the mutant Htt protein with elongated polyQ.
Defined amount of recombinat wild-type Htt (25Q) and mutant Htt (46) were analyzed by time resolved Förster resonance energy transfer using the antibody pair 4C9 & 2166, which recognized equaly well mutant Htt and wild-type Htt.
A: Time-resolved FRET detection of Htt exon1 with 200Q in brains of R6/2 mice. Wild-type mice served as negative controls. The relative concentration of Htt measured by the 2B7 & MW1 antibody pair decreased significantly in 12-week-old symptomatic mice when compared to 4-week-old presymptomatic mice. B: Brain Htt aggregates in R6/2 mice were determined by AGERA (same samples analyzed in A). As expected, a significant increase in aggregate load was found in the older mouse group. C: Specific detection of soluble Htt with the 2B7 & MW1 antibody pair. R6/2 brain homogenates were separated in soluble and insoluble material by ultracentrifugation. The main pool of insoluble Htt aggregates measured by AGERA was recovered in the pellet. In contrast, time-resolved FRET predominantly revealed soluble Htt in the supernatant. D: Detection of Htt in R6/2 tissues. Cortex and muscle samples from 6-week-old R6/2 mice contained significant amounts of Htt when compared to wild-type siblings (n=3). Mutant Htt was detected also in plasma and CSF from 9 to 12-week-old R6/2 mice (n=9) when compared to wild-type mice (n=4). E: Full-length Htt detection in different brain regions of knock-in Hdh140 mice (n=3) expressing Htt with 140Q at endogenous levels. Wild-type siblings (n=3) were used as negative controls. All data average values with standard deviations.
A: Representative example of an AGERA blot of brain samples from R6/2 or wild-type (WT) mice at 4 and 12 weeks of age. An age-dependent increase in Htt aggregate load is evident in the R6/2 mouse brain. B: R6/2 brain homogenates of 4 and 12-week-old animals before (Start) and after separation by ultracentrifugation in soluble (Super.) and insoluble (Pellet) material, were analyzed by AGERA. Aggregates are efficiently recovered in the insoluble fraction. No aggregates were detected in the supernatant fractions.
The present invention relates to bioassays for polyQ protein associated with disease and their use as diagnostic tools, for monitoring disease progression or for monitoring the efficacy of treatment of the disease.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term “mutated polyQ protein” or “polyQ protein” refers to the form of a the polyQ protein containing a polyglutamine expansion associated with the development of disease. For example “mutated polyQ huntingtin” refers to the huntingtin with a polyglutamine expansion beyond 36 glutamines and associated with Huntington's Disease.
The term “single step assay”, refers to an immunoassay in which no separation or washing is necessary and usually can be run after a single biochemical handling.
The term “posttranslational modifications”, refers to cellular modifications of the expressed protein such as proteolytical cleavage, phosphorylation, acetylation, ubiquitination SUMOylation or other covalent modifications of the polypeptide backbone.
The polyglutamine diseases for which the bioassay of the present invention is useful is listed in the following table.
The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i. e., “antigen-binding portion”) or single chains thereof. A naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
The term “antigen-binding portion” of an antibody (or simply “antigen portion”), as used herein, refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., huntingtin). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding region” of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
As used herein, an antibody that “specifically binds to a polyQ protein is intended to refer to an antibody that binds to the polyQ-protein with a KD of 1×10−8 M or less, 1×10−9 M or less, or 1×10−10 M or less. An antibody that “cross-reacts with an antigen other than the polyQ-protein” is intended to refer to an antibody that binds that antigen with a KD of 0.5×10−8 M or less, 5×10−9 M or less, or 2×10−9 M or less. An antibody that “does not cross-react with a particular antigen” is intended to refer to an antibody that binds to that antigen, with a KD of 1.5×10−8 M or greater, or a KD of 5-10×10−8 M or 1×10−7 M or greater. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding against these proteins in standard binding assays.
The term “Kassoc” or “Ka”, as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “Kdis” or “KD,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “KD”, as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A method for determining the KD of an antibody is by using surface plasmon resonance, or using a biosensor syttem such as a Biacore® system.
As used herein, the term “Affinity” refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
As used herein, the term “Avidity” refers to an informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody epitope affinity; the valence of both the antigen and antibody; and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope.
As used herein, the term “cross-reactivity” refers to an antibody or population of antibodies binding to epitopes on other antigens. This can be caused either by low avidity or specificity of the antibody or by multiple distinct antigens having identical or very similar epitopes. Cross reactivity is sometimes desirable when one wants general binding to a related group of antigens or when attempting cross-species labeling when the antigen epitope sequence is not highly conserved in evolution.
As used herein, the term “high affinity” for an IgG antibody refers to an antibody having a KD of 10−8 M or less, 10−9 M or less, or 10−10 M or less for a target antigen. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype refers to an antibody having a KD of 10−7 M or less, or 10−8 M or less.
As used herein, the term “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles, etc.
Various aspects of the invention are described in further detail in the following subsections.
Time Resolved Förster Resonance Energy Transfer
Time resolved Förster Resonance Energy Transfer (time resolved FRET) is a preferred detection technology for use in a bioassay of the present invention. The technology that has been available for monitoring biomolecular interactions since the early 1990's (Mathis, 1993). There are many different applications of this technology utilizing several aspects of the fluorescence characteristics of lanthanide ions. The large Förster's distance of the rare earth ions is up to 9 nm which is much larger than for many fluorescent compounds which have Förster's distances of between 1-7 nm. The effect of this larger distance is that it is possible to transfer absorbed energy over much longer distances than it is possible for many Förster resonance energy transfer pairs. This then makes it possible to use rare earth chelates as generic immunodetection reagents (Bazin et al., 2001). The second advantage that rare earth Förster resonance energy transfer pairs have is that the time it takes for the fluorescence to decay is greatly delayed thus allowing time resolved fluorescence. The effect of this is to reduce the influence of background fluorescence from small molecules being tested. The ability to monitor ratiometric readouts allows the possibility to correct for liquid dispensing errors, thus helping to reduce assay variability and improve data quality (Imbert et al., 2007).
In time resolved Förster resonance energy transfer (
In addition to the Förster resonance energy transfer pair Eu3+-cryptate+D2, a second labeling pair has been tested, whereby Lumi4™-Tb, a terbium complex developed by Lumiphore Inc., is the new donor in the time-resolved Förster resonance energy transfer assay. The emission of Tb2+-cryptate is different from Eu3+-cryptate and thus a green acceptor can be used (Alexa488, fluoresceine). Thus, it is now possible to measure two different proteins (or different combination of epitope, e.g. for analyzing post-translational modifications) in the same sample. The emission of Tb2+-cryptate is different from Eu3+-cryptate and thus in addition to the red D2 acceptor, a green acceptor can be used (Alexa488, fluoresceine).
Another preferred detection technology is electrochemiluminescence detection, e.g. as commercially available from Meso Scale Discovery, 9238 Gaither Road, Gaithersburg, Md. 20877, USA.
Meso Scale Discovery (MSD) Technology's electrochemiluminescence detection technology uses SULFO-TAG labels that emit light upon electrochemical stimulation initiated at the electrode surface of microplates. Multiple acceptor antibodies can be spotted in the same well thus allowing detection of up to 10 different proteins in the same sample, bound huntingtin (wild-type and mutant) is detected by a pan anti-huntingtin antibody.
We spotted two capture antibodies Abeta42 (against the correspondingly tagged wild-type 25Q-huntingtin) and Abeta40 (for mutant 72Q-huntingtin) on a 4 spot/well plate. Only samples containing lysates from cells with induced expression of wild-type 25Q-huntingtin results in a signal when the anti-Abeta42 spot is analyzed. In contrast, the samples containing lysates from cells with induced expression of mutant 72Q-huntingtin are positive in the anti-Abeta40 spots. Htt bound to the plates was detected with the biotinylated Nov1 antibody. The result is shown in
Thus, theoretically, up to 10 different huntingtin forms (mutant, wild-type, specific post-translational modifications) can be detected in the same sample assuming selective antibodies are available.
Assay Description
Time resolved Förster resonance energy transfer has been used to monitor a number of different biological analytes such as small molecules (e.g. cAMP (Gabriel et al., 2003)) as well as small secreted cytokines (e.g. IL-8 (Achard et al., 2003)) as well as the levels of phosphorylated proteins in in vitro assays (Riddle et al., 2006). There have also been reports of using time resolved Förster resonance energy transfer to monitor the levels of phosphorylated proteins in cell lysates using cell lines over expressing protein substrates of interest. Here we extent these observations by designing combinations of antibodies that give optimal time resolved Förster resonance energy transfer signals and allow the detection of protein expressed at endogenous levels.
Technology Development
Time resolved Förster resonance energy transfer detection of amyloid β peptide in fluid biological milieu has been previously described (Clarke and Shearman, 2000) and is currently commercially available. The amyloid assay takes advantage of high-affinity antibodies directed against two well characterized epitopes in the amyloid β peptide. We designed a library of small peptides which carry these epitopes (
Protein Detection and Signal Specificity
The neuronal clonal cell lines created express the tagged polyQ-Htt and wild type Htt at endogeneous levels upon full induction with no detectable basal expression as shown by western blot (FIG. 2A,B). Expression levels of the constructs after induction are stable over time (
Next, we adapted the assay to a 1536 microwell format. For this, further work was performed using 573-Q72 expressing clone with 2B7-K and β1-D2 antibody combination in order to quantify uncleaved polyQ-Htt-protein. One of the advantages of using a ratiometric readout as used in the time resolved Förster resonance energy transfer method is that adaptation to a miniaturized assay format is readily facilitated because the assay signal is not dependent on the path length of the detection system or the absolute number of particles being detected. In addition ratiometric readout are also more robust to errors in liquid handling again facilitating assay miniaturization. After miniaturizing the format to cells grown directly in a 1536 microwell plate, assay protocol was optimized for lysis buffer (
Time-Resolved Förster Resonance Energy Transfer Assay for Detection of Endogenous Huntingtin.
Amino-terminal fragments of mutant Htt are neurotoxic in vitro and in vivo and are thought to cause Huntington's disease (Arrasate et al., 2004; Li et al., 2000; Varma et al., 2007). Mutant Htt toxicity and aggregation are dependent on polyQ length, Htt fragment length, and level of mutant Htt expression (Colby et al., 2006; King et al., 2008; Machida et al., 2006; Scherzinger et al., 1999; Wang et al., 2005). A critical step towards a disease-modifying treatment or cure for HD is the ability to detect changes in mutant Htt protein levels in the presence of therapeutic modalities in a simple one-step assay. Furthermore, sensitive and efficient Htt measurement may represent a biomarker to analyze the clinical onset or progression of HD. We recently demonstrated the feasibility to measure in one step intracellular mutant Htt using a time resolved-Förster resonance energy transfer assay. In this assay, an antibody pair recognizes a short artificial tag fused to a Htt fragment. (Paganetti et al. 2009)) (
There is general agreement that Htt toxicity is centered at the amino-terminus of Htt protein, therefore we developed a highly sensitive assay for endogenous mutant Htt using amino-terminal-specific antibodies. The monoclonal antibody 2B7 was conceived as a pan antibody for measuring fragments and full-length mutant Htt and binds to the 17 amino acids immediately amino-terminal to the polyQ-repeat of Htt. When applied in combination with MW1, a polyQ-binding antibody, the 2B7 & MW1 pair specifically detected a 573 amino acid long fragment of wild-type (25Q) and mutant (72Q) Htt expressed in HN10 cells (
A precise comparison of the polyQ-dependent detection of wild-type and mutant Htt in different HN10 cell lines is hampered by clone to clone variations in protein expression levels. As an alternative, we opted for a lentiviral approach for expression of untagged Htt-constructs with 25 or 72 glutamines in a homogenous population of mouse embryonic stem cells (ESC; (Bibel et al., 2004)). A predefined virus titer led to equal expression of the constructs as demonstrated by western blot. Signal strength in time resolved-Förster resonance energy transfer increased with polyQ-length (
For evaluating the use of the time resolved-Förster resonance energy transfer assay to detect endogenous, full-length Htt, we selected ESC with the Htt gene deleted (Htt knock-out as negative control) or modified by a polyQ insertion (polyQ knock-in as positive control). A significant signal was obtained in samples from the 140Q knock-in ESC when compared to samples from the Htt knock-out ESC (
Simultaneous Detection of Two Proteins by Duplex Time Resolved Förster Resonance Energy Transfer
One additional application is the duplex determination of tamed wild-type and mutant Htt573 in the same sample, e.g. in HN10 cell lysates expressing both 25QHtt573 and 72QHtt573 whereby the human sequence of Htt was truncated at amino acid 573 and wild-type Htt (25Q) was tagged with the beta1 and 32A7 antigens and mutant Htt (72Q) was tagged with beta1 and 25H10 antigens (
Production and Purification of Recombinant Human Huntingtin
A cDNA encoding for a fragment of human Htt 573 amino acid long (with 25 glutamines and truncated after amino acids ThrThrThrGluGlyPro*) was subcloned downstream of Glutathione S-transferase and the vector transfected in E. coli. Bacterial cultures were grown at 37° C. to an OD600 of 1, cooled on ice, isopropyl β-D-1-thiogalactopyranoside was added and cultures were incubated at 12° C. for 18 h. Bacteria were collected by centrifugation and lysed by sonication with phosphate buffered saline and 1% Tween-20. Lysates were cleared by centrifugation and incubated with 2 ml/10 ml lysate Glutathione resin. Beads were washed twice with PBS/0.5% Tween-20 and twice with cleavage buffer (50 mM Tris pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween-20, each wash 25× bed volumes). Beads were incubated for 16 h at 4° C. on a rotator with one bed volume cleavage buffer containing 40 U/ml PreScission Protease. The supernatant containing purified Htt protein was recovered and the beads were washed with another bed volume of cleavage buffer (
Simultaneous Detection of Two Htt Isoforms by Duplex Time Resolved Förster Resonance Energy Transfer
A second possible application is to study different isoforms of Htt using additional antibodies specific for Htt as listed, but not limited, to the antibodies in the table below:
MW1 and MW8 were generated by Prof. Patterson, Caltech (Ko and Patterson, 2001). 2166 is a commercially available antibody (Chemicon), all other antibodes were generated by our laboratories.
To analyze the specificity of the different antibody pairs, we used purified recombinant Htt protein expressed in bacteria as a standard to calibrate the bioassay and duplex time resolved Förster resonance energy transfer.
In the first application, we used the two antibody pair 2B7 & MW1 and 2B7&4C9 for the detection of recombinant Htt proteins with 25Q and 46Q. We observed that while 2B7 & MW1 (Alexa488 channel) recognized better mutant Htt in a polyQ-dependent manner, 2B7 & 4C9 (D2 channel) resulted in a stronger signal for wild-type Htt than for the mutant Htt protein with elongated polyQ (
Further validating this conclusion, we observed that when using the antibody combination 4C9 & 2166, which recognize two epitpes down-stream of the polyQ stretch, both wild-type Htt (25Q) and mutant Htt (46Q) were detected equally well (
Detection of Soluble Mutant Htt in Central and Peripheral Tissues of Murine HD Models and Significant Changes in Soluble Brain Htt as a Function of Disease Progression.
The single-step bioassay for Htt was next used to analyze brain homogenates obtained from 4 and 12 week-old R6/2 mice and aged-matched wild-type mice. R6/2 mice develop an aggressive HD-like phenotype because of the ubiquitous expression of mutant Htt exon1 driven by the human Htt promoter (Mangiarini et al., 1996).
We extended our analysis to include muscle and plasma samples from 6 week old as well as corticospinal fluid samples from 9 to 12 old R6/2 or WT mice. We found significant detectable amounts of mutant Htt in the R6/2 mice when compared to their normal siblings in all tissue samples analyzed, although the signals were significantly lower than those detected in cortical extracts (
In the context of a bioassay for mutant Htt, the R6/2 mouse model of HD based on the expression of a short fragment of Htt may have only a limited value as a model of the human situation in which mutated full-length Htt is expressed. As an alternative, we applied the time resolved-Förster resonance energy transfer assay for the analysis of mutant Htt in a knock-in mouse model expressing endogenous mouse full-length Htt with a polyQ-stretch of 140 glutamines (Menalled et al., 2003). Similar to the R6/2 mouse samples, significant amounts of mutant Htt were detected in every brain area analyzed as well as in full blood samples (
Sensitive and PolyQ-Dependent Huntingtin Detection in Human Tissue Samples.
As the sensitivity and specificity of the mutant Htt bioassay was clearly demonstrated in mouse tissue samples, we next turned our attention to detection of endogenous mutant Htt in human post-mortem cortex tissue obtained from three healthy volunteer (HV) controls and three HD brains. In a first experiment, the Htt bioassay measured a 2.7-fold higher level of Htt (p<0.001) in all three HD patients compared to the HV controls (
As in most clinical settings it is much easier to obtain blood samples, we then tested three different blood fractions obtained from five living HD patients and four HV subjects. In a blinded experiment, it was possible to unambiguously distinguish the HD patient samples from the four control samples in whole blood, isolated erythrocytes and buffy coats. The relative amount of Htt determined by the time-resolved FRET assay was z-transformed to allow score comparison between the blood fractions (
Towards routine use for clinical research projects, the assay was used for the analysis of a large group of buffy coats PBMC samples from healthy volunteers and HD patients (kindly provided by Sarah Tabrizi): 100 subjects with two time points for each subject. In this double blinded study, when using a signal threshold established by an earlier experiment using samples provided by Steven Hersch, we correctly assigned 43 our of 44 samples as belonging to the healthy controls. Thus, the assay performs exceptionally well to separate healthy from HD using a blood fraction as starting material. The results are shown in
We have developed a bioassay for determination of soluble mutant Htt and demonstrated its use to measure mutant Htt levels in cell lysates, animal and human tissues.
The time resolved-Förster resonance energy transfer assay is a simple, one-step methodology that requires only small sample volumes. Quantitative determination of mutant Htt levels are therefore possible with as little as 5 μl human full blood, providing the possibility to determine soluble mutant Htt levels multiple times over a longer clinical trial period without affecting the patient as obtaining the sample is minimally invasive. In addition, the ability to correct for artifacts using the time resolved-Förster resonance energy transfer method allows for a very reliable quantification of mutant Htt even in small sample sizes (Imbert et al., 2007).
To verify the specifity of our detection method for soluble mutant Htt, several experimental steps were taken. Importantly, we based our bioassay on a method that has been recently described to detect intracellular levels of tagged Htt fragments in a sensitive, robust and reliable manner as indicated by a high Z-factor value, a common statistical parameter that reflects assay quality in terms of reliability and robustness (Zhang et al., 1999), (Weiss et al. submitted). By exchanging the detection antibodies of the high-throughput screen to an antibody pair that recognizes endogenous Htt epitopes, we were able to show detection of untagged Htt fragments in a stable neuronal cell line with inducible expression of tagged Htt. In order to specifically detect mutant Htt levels over wild-type Htt, one of the antibodies is directed against the polyQ-repeat that is elongated in Huntington's Disease. We then showed that the signal intensity directly correlates with the polyQ length in lentiviral infected embryonic stem cells as well as in cell lysates obtained from wild-type and polyQ-knock-in embryonic stem cells and embryonic stem cell derived neurons. Critically, using knock-out embryonic stem cell lysates void of any Htt protein expression, we were able to prove the Htt specificity of our signal. As the detection method is polyQ-length dependent, it should be noted that while wild-type Htt is not detected in murine cells or animal tissue due to the WT-polyQ-length of only 7 glutamines, human healthy polyQ length normally resides around ˜20 glutamines (Myers, 2004), a length that is also detected by our method. However, the intensity of this healthy Htt derived signal only contributes little to the total signal that is largely comprised of mutant polyQ Htt detection with polyQ-length >39 glutamines.
We proceeded to analyze mouse models of HD and for the first time were able to quantifiably determine soluble, non-aggregated mutant Htt levels in various tissue samples of two different HD mouse models. Notably, we found that the levels of soluble mutant Htt decrease while the amount of insoluble Htt aggregates increase in the brains of aging R6/2 mice. This decrease of an aggregation prone monomeric species upon aging resembles similar findings in Alzheimer Disease whereby a decrease in Abeta42 levels is a marker for disease progression (likely to be caused by recruitment of soluble Abeta as a function of increased plaque burden).
Finally, we tested human post-mortem cortex samples as well as full blood and blood-derived human samples from living control and HD patients. We were able to clearly distinguish between healthy and HD patient samples by the intensity of the signal alone. This quantitative detection of soluble Htt in readily available human tissue sample opens up the possibility to determine the value of this soluble mutant Htt quantification for use as a potential biomarker for HD disease progression. In that regard, it is interesting that the signal measured in buffy coat fraction trends to correlate with severity of disease progression as determined by the total function capacity score in the analyzed HD patients. This trend was not observed in full blood or erythrocytes. However, erythrocytes represent the vast majority of cells found in full blood and since erythrocytes display a shorter lifespan than some of the lymphocytes found in the buffy coat fraction, this difference could be due to the longer lifespan of a lymphocytes subpopulation in which effects of mutant huntingtin monomer expression, e.g. huntingtin aggregation, can accumulate over time, leading to a decreased signal of soluble mutant huntingtin similar to what we observed in R6/2 mice with advanced disease progression. Further longitudinal studies with a larger HD patient population could help to elucidate this intriguing possibility. In addition, since potential HD therapies could be aimed at influencing the soluble mutant Htt pool directly (e.g. compounds that alter aggregation, compounds that act on the chaperone system or compounds that act on autophagy) the precise quantification of soluble mutant Htt could also find application as a marker for treatment success in human clinical trials.
In summary, our bioassay is a very simple, one-step methodology that requires small sample volumes. In addition, the artifact corrected nature of time resolved-Förster resonance energy transfer allows for very reliable Htt-quantification with a single small sample per subject, making the method useful for experiments that are limited by sample numbers or sample volume as it is often found in human clinical trials. Because signal specifity of the method depends on the antibody pair used, the method could also find further application not only for HD but also for other diseases, especially other polyQ-diseases like the spinocerebellar ataxias.
An interesting development of our assay is multiplexing and thus allow to establish relative measurements for several huntingtin forms. We have demonstrated that this is possible for mutant versus normal huntingtin, but it will be very interesting to develop additional antibody combinations to measure the extend of post-translational modifications relative to normal huntingtin such as proteolytical cleavage, phosphorylation, acetylation, ubiquitination SUMOylation and other naturally occurring covalent modifications of the polypeptide backbone.
The sample material for use in an assay according to the present invention may be human body fluids such as urine, saliva, plasma, serum and corticospinal fluid as well as tissue extracts from organs such as brain, muscle, skin, hair, blood cells and other central and peripheral human organs. Tissue extracts can be obtained by homogenization or detergent lysis of tissue biopsies
When the bioassay of the present invention is used as a diagnostic tool any of the human samples described above can be analysed and compared to those obtained from healthy volunteer controls.
When the bioassay of the present invention is used for monitoring disease progression any of the human samples described above can be analyzed longitudinally as a function of disease progression or before and after phenoconversion
When the bioassay of the present invention is used for monitoring the efficacy of treatment of the disease any of the human samples described above can be analyzed before and after pharmacological treatment
Methods & Materials
Generation of Peptides and Antibodies
Peptides comprising epitopes, against which antibodies 25H10, 32A7 or β1 are reactive, and which are separated by different linker sequences, were custom produced by MIT biopolymers laboratory. Amyloid β40 peptide was purchased from Bachem (Bubendorf, Switzerland).
25H10 antibody specific against GGVV-epitope, 32A7 antibody specific against VVIA and β1 antibody directed against EFRH are described elsewhere (Paganetti et al., 1996). 2B7 antibody was custom designed against the first 17 amino acids of Htt protein (GENOVAC, Freiburg, Germany). MW1 antibody specific against the polyglutamine stretch of Htt and developed by Dr. Paul Patterson were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa 52242. Custom europium cryptate and D2-fluorophore labeling of the antibodies were performed by CisBio (Bagnols/Ceze, France). Depending on the batch used, antibodies were cross-linked to 5 to 7 mol europium cryptate or D2-fluorophore per mol antibody.
Generation of Neuronal Cell Lines
Neuronal HN10 cells (Lee et al., 1990) were used to create inducible clones with expression of 573-Q25 and/or 573-Q72 Htt amino-terminal. In short, cells were transfected with the rheoswitch receptor plasmid (New England Biolabs) and cultured under selection of 1 mg/ml G418 (Invitrogen). Clones were screened for cell morphology, transfected with inducible luciferase reporter construct and induced for 2 days. Clone with best induction ratio were selected and used for subsequent transfection with 573-Q25 or 573-Q72 inducible plasmid. After selection with 1 mg/ml G418 and 1 mg/ml Hygromycin (Invitrogen), inducible expression of Htt fragments in the clonal lines were monitored with herein described time resolved Förster resonance energy transfer detection method and clones with no basal expression and highest inducible expression were chosen for use in assay format.
Other Cellular Models.
The knock-in embryonic stem cells (ES cells) were generated as described in (Wheeler et al., 1999; White et al., 1997). The neomycin selection cassette was removed by a second electroporation with a plasmid expressing cre recombinase. Embryonic stem cell-derived neurons (ES neurons) were generated using the differentiation protocol as published by (Bibel et al., 2007; Bibel et al., 2004). In brief, ES cells were cultivated on mitomycine-inactivated mouse embryonic fibroblasts for at least two passages after thawing in ES medium containing 15% foetal calf serum (FCS) and 1000 U/ml LIF (leukemia inducing factor). Subsequently, they were cultured without fibroblast feeder cells for two more passages. Embryoid bodies (EBs) were formed on bacterial dishes in EB medium containing 10% FCS but no LIF and incubated for 8 days with the addition of retinoic acid on the last four days. EBs were dissociated by trypsinisation and plated on poly-l-lysine and laminin coated plates in N2 medium and changed to neuronal differentiation medium as described by (Brewer and Cotman, 1989) two days after dissociation.
Detection of Peptides by Time Resolved Förster Resonance Energy Transfer
Peptides were prediluted in DMSO to 800 μg/ml. DMSO solutions were further diluted in ⅕ RIPA buffer to 3 ng/ml final concentration. 3 ng/ml amyloid β40 peptide was used as control. 10 μl peptide solution per low-volume 96-well were mixed with 5 μl of antibody solution (beta1-D2 20 ng/well, 25H10-K 2 ng/well in 50 mM NaH2Pa4, 400 mM NaF, 0.1% BSA, 0.05% tween) and incubated at 4° C. overnight. 620 and 665 nm signals were measured with a RUBYstar (BMG Labtech) reader.
96 Well Format
20.000 cells/well were seeded in 100 μl normal grow medium (DMEM (Gibco), 10% FCS, penicillin and streptomycin). After 2 h medium was removed and 200 μl inducing medium (normal growth medium plus inducing ligand) was added to start expression of Htt fragments. After 3 days, medium was removed and 30 μl/well readout buffer (20 μl of different lysis buffers and 10 μl of β1-D2 and 25H10-K or 32A7-K in 50 mM NaH2Po4, 400 mM NaF, 0.1% BSA, 0.05% tween) was added. After incubating 30 min incubation at room temperature, lysates were transferred to low volume black bottom 96 well plate. After 3 h at 4° C. 620 and 665 nm signals were measured. with a RUBYstar (BMG Labtech) reader.
1536 Well HTS Miniaturization and Compound Screen
573-Q72 expressing clone was incubated for 72 hours at 37° C., 5% CO2 with inducing medium to facilitate expression of polyQ-htt construct. 3 μl of a 2000 cells/μl cell suspension were then seeded per well in a 1536-microtiterplate (Greiner) and incubated overnight −/+ compound treatment. 3 μl of Lysis buffer (1× PBS+1% Triton X-100, complete protease inhibitors) were added and incubated for 30 min at room temperature. 2 μl antibody dilution in 50 mM NaH2PO4, 400 mM NaF, 0.1% BSA, 0.05% tween was added to a final dilution of 60 pg/well europium labeled antibody and 800 pg/well D2-labeled antibody. Plates were incubated at room temperature as indicated. Measurements were performed with a View Lux machine with the following settings: Label 1 time resolved Förster resonance energy transfer_Eu-K_(E:800K,Xsec,BF4, GN:high,SP:slow), Label 2 time resolved Förster resonance energy transfer_XL665_(E:800K,Xsec,BF4, GN:high,SP:slow)
Animal Models.
Heterozygous transgenic R6/2 males of CBAxC57BL/6 strain were obtained from G. Bates laboratory (Mangiarini et al., 1996) and bred with CBAxC57BL/6 F1 females. The offspring were genotyped by PCR assay of DNA obtained from tail tissue. The animals were housed in a temperature-controlled room that was maintained on a 12 hr light/dark cycle. Food and water were available ad libitum. All experiments were carried out in accordance with authorization guidelines for the care and use of laboratory animals.
For time resolved Förster resonance energy transfer assay detection of huntingtin, 2-3 months old animals were anesthetized with 3-5% isofluran followed by an intraperitoneal dose of 100 mg/kg Ketamin and 10 mg/kg xylazine. After CSF and blood collection, animals were given a sodium pentobarbital overdose (150 mg/kg). Muscle (gastrocnemius) and brain were immediately further collected for Förster resonance energy transfer analysis. Brain homogenate loaded 100 ug per well (10 mg/ml protein concentration)
Aggregate Analysis (AGERA).
AGERA analysis was performed as described (Weiss et al., 2008). In short, R6/2 brains were homogenized in 10 volumes (w/v) PBS+0,4% TritonX100 and Complete Protease Inhibitor (Roche Diagnostics). Brain samples corresponding to 0.15 □g of total protein were loaded per AGERA lane. For separation of brain homogenates into soluble and non-soluble fractions, homogenates were centrifuged at 124 000 g for 1 h, supernatant was aliquoted (soluble fraction) and pellet was resuspend in equal to starting volume PBS+0.4% TritonX100 (non-soluble fraction).
BioAssay
Brain and muscle tissue were homogenized in 10× volume sample buffer (PBS+1% Triton X-100+ compleate protease inhibitor). Blood, plasma and corticospinal fluid samples were prediluted 1:1 in sample buffer. 10 μl sample and 5 μl antibody dilution (europium cryptate and D2 labeled antibodies in 50 mM NaH2PO4, 400 mM NaF, 0.1% BSA, 0.05% Tween) was added to each well to a final dilution of 1.5 ng/well 2B7-europium labeled antibody and 30 ng/well MW1-D2-labeled antibody. Plates were incubated at 4° C. for 1 h. Measurements were performed with a Xenon-lamp Envision Reader for 620 and 665 nm wavelengths after excitation at 320 nm (time delay 100 μs, window 400 μs, 100 flashes per well).
Data Analysis
Time resolved Förster resonance energy transfer measurement results in two different signals. 620 nm signal from the europium cryptate labeled antibody can be used as an internal reference for possible interfering artifacts of the assay such as signal quenching or absorption by compounds, sample turbidity as well as differences in excitation energy or sample volume. 665 nm signal results from D2 labeled antibody which is excited by time resolved energy transfer from the europium cryptate. The calculated 665/620 nm ratio therefore is an artifact corrected specific signal of the two bound antibodies to their antigen and hence a precise reflection of the amount of antigen present in the sample. For 96 well data time resolved Förster resonance energy transfer signals are given as the ratio between those two wavelengths:
Ratio665/620induced−Ratio665/620noninduced*10000
For 1536 microtiter well optimization data, time resolved signals are presented as ΔF values, a format more suitable to take day-to-day assay variations into account as it is a background correctd value:
ΔF=(Ratio665/620induced−Ratio665/620noninduced)/Ratio665/620noninduced*100
Analysis of high throughput screening data was conducted using an inhouse data analysis software, this software is able to normalize activity to % remaining activity with the use of high and low control samples present on a plate and to correct plate effects using a local regression algorithm that corrects for local plate effects (Gubler, 2006). Z-factor was calculated according to (Zhang et al., 1999).
Statistical Analysis
Quantification of cellular and mouse values are presented as averages with standard deviations. Significances were calculated by students' t-test.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 08161736.7 | Aug 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/060013 | 8/3/2009 | WO | 00 | 2/4/2011 |
